| Porcine Epidemic Diarrhea?PED?is a highly contacting intestinal infectious disease caused by Porcine Epidemic Diarrhea Virus?PEDV?infecting pigs,which was characterized by vomiting,acute watery diarrhea,dehydration and weight loss.Since the outbreak of the PED in southern China at the end of 2010,PEDV has rapidly swept through various provinces and cities in China,causing huge economic losses to the pig industry.To know deeply the current spread trend,genetic variation and molecular evolution of PEDV in China,the suspected diarrhea samples collected from the end of2016 to the end of 2018 were detected and analyzed,and then S genes and complete genomes of some strains were sequenced.Subsequently,genetic variation and molecular evolution were analyzed.The specific results are as follows:1.The current situation of PEDV in China.In this study,we collected a total of 898 intestinal tissue and fecal samples from different pig farms in Hubei,Shandong,Henan,Hunan,Zhejiang,Guangdong and other18 provinces during from the end of 2016 to the end of 2018,including 636 intestinal tissue samples and 262 fecal samples.The results showed that 232 samples of intestinal tissue were PEDV positive,the positive detection rate was 36.48%,81 fecal samples were PEDV positive,the positive detection rate was 30.92%.The overall positive rate of PEDV was 34.86%.Some strong positive samples from different farms were selected for sequencing.S full-length gene of 76 strains of and complete genome of 9 strains were sequenced.According to the difference of sequence identity of S gene in different provinces,only one sequence was retained and the others with more than 99%sequence identity were removed.Finally,45 S gene sequences were screened for phylogenetic analysis.Based on 45 strains of PEDV S gene,the NJ phylogenetic tree was constructed.According to the phylogenetic tree analysis of S gene construction,PEDV can be divided into G1 type and G2 type,and can be subdivided into G1-a subtype?classic strains and its source vaccine strains?,G1-b subtype?S-INDEL strains?,G2-a Subtype?variant strains?,G2-b subtype?recombinant strains?.The results showed that 44 strains were G2-a subtype?variant strains?and 1 strain were G1 type among 45 strains detected in this study.It was indicated that the current epidemic strains of PEDV in China are still mainly G2-a subtype variant strains.According to the genetic relationship of S gene of 44 strains of G2-a subtype PEDV,the epidemic strains of G2a PEDV could be further subdivided into3 subtypes,mainly G2-a1 subtype and G2-a3 subtype.Among them,17 sequenced strains such as E3-SX2017,V1-HB2018 and E6-SN2017 were classified as G2-a1 subtypes,which were closer to XM2-4 and HBXY1 in terms of their phylogenetic relationship.The sequence identity was between 95.7%and 99.5%;And 23 sequenced strains such as M3-SX2017,G2-HE2017 and Z7-HA2018 were G2-a3 subtypes,which were more similar to AJ1102 in terms of their genetic relationship.The sequence identity was between 95.8%and 99.6%.2.Diversity of genetic variation of PEDV S gene.In this study,PEDV S gene of 45 strains were compared with that of variant strain AJ1102,classical strain CV777,LZC,attenuated vaccine strain attenuated CV777,S-INDEL strain ZL29,cell subculture adaptive strain YN144,natural attenuated strain FL2013 and recombinant strain CH/HNQX-3/14.In terms of amino acids insertion and deletion,G7-SC2017,W3-AH2018 and Y9-SX2018 had only two amino acids insetions at 59-62aa,which was different from that of most G2-a subtype strains.At the same time,it was found that the S protein of 21 G2-a subtype strains was mutated in the 1199th amino acid of the S protein C-terminus,while the S protein of 23 G2-a subtype strains were deleted in the site.In the aspect of S protein epitopes,SS6 and COE epitopes were susceptible to mutation.The 526th amino acid of COE epitope was the most complex,which can be mutated to H/Y/R/N from L;The conserved epitopes SS2 and 2C10 also had sporadic amino acid mutations.V7-HB2018 was the only G1 strain detected in this study.Although its S protein was very similar to the known S protein of S-INDEL strain,which showed similar amino acid insertions and deletions.However,there were some differences in the gene level of S protein,and the sequence identity was only 97.0%-97.5%.The complete genome was sequenced.Further bioinformatics recombination analysed showed that it was a new recombinant S-INDEL strain which parent strains were CH/ZMDZY/11 and CV777,and its breakpoint was from 20721nt to 21341nt analyzed by Simplot.3.Analysis of its evolution based on PEDV genome.By referencing the complete genome sequence of PEDV CV777 and AJ1102,17pairs of primers were designed to amplify the complete genome sequence of PEDV.And a total of 8 strains of G2 type PEDV genome and 1 strain of G1 type PEDV genome?V7-HB2018?were amplified and sequenced.58 genome sequences of G2 type PEDV in China and 1 genome sequence of G2 type PEDV in Korea were obtained from GenBank database.The MCC phylogenetic tree was constructed by BEAST.It can be seen that the base substitution rate of G2 type PEDV in China is 1.0487×10-3 times/site/year,and its time to the most recent common ancestor?tMRCA?was 26.4059 years ago?that is 1991?.Based on Bayesian Skyline Plot analysis,the effective population size of G2 type PEDV in China has experienced two expansion periods.the most recent period was from 2010 to2012,which was consistent with the fact of PED outbreak in China at the end of 2010.The positive selection sites analysis of structural genes of G2 type PEDV strains in China was analyzed by DataMonkey.It was found that there were three positive selection sites on S protein and one positive selection site on N protein,but no related positive selection sites were found on M protein and E protein.The three positive selection sites on the S protein are located on the overlapping area of S1-NTD and S1-CTD,S1-CTD and COE epitope,respectively.The adaptive mutations on these three positive selection sites may increase the infectivity of the virus and escape host immunity,so that it can constantly adapt to the new environment. |
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